Abstract - NO CHANGES FROM ORIGINAL Microtubules (MTs) exhibit dynamic instability in which they exist in growing, pausing, or shrinking states that interconvert stochastically. This behavior provides the mechanism by which microtubules assemble into a seemingly infinite variety of structures that provide countless cellular functions including cell motility, mitosis, and axonal transport. Numerous microtubule-associated proteins (MAPs) and motors bind to the microtubule lattice or to the growth-favored ?plus? end to regulate microtubule assembly dynamics, organization, and interactions. Important questions that are still unanswered are: how the ensemble behavior of these proteins collectively regulates microtubule dynamics and how these activities are regulated through cell-cycle stages and in different cellular subcellular-compartments. A biochemical cell-extract assay recently developed in the Barnes laboratory unifies, for the first time, two of the most powerful approaches for studies of microtubule dynamics: biochemical extract studies and genetics. Cell extracts are made from budding yeast mutants and dynamics of single microtubules are observed. Since mitosis is a highly conserved process, lessons learned from these studies are likely to apply generally. Unlike many other assays, this assay uses homologous sources of tubulin and MAPs, avoiding species incompatibility. Moreover, dynamics of single microtubules are quantitatively analyzed by highly sensitive Total Internal Fluorescence Microscopy. The three aims are: (1) To determine how specific MAPs and motors affect microtubule assembly dynamics and to reveal emergent properties that arise from their combined activities. Extracts will be prepared from wild-type yeast and mutants of different microtubule dynamics regulators, singly or in combinations, and microtubule dynamics in the extracts will be quantitatively analyzed to parse the contributions of individual proteins to collective microtubule dynamics regulation. Using cell-cycle-staged extracts from mutants of MT dynamics regulators, proteins responsible for programmed changes in microtubule dynamics through the cell cycle will be identified. (2) To determine how activities of these proteins are coordinately regulated through the cell cycle. For four different cell-cycle stages, phosphorylation sites on MT dynamics regulators will be mapped by mass spectrometry of the regulators. Functional importance of the identified cell-cycle-specific phosphorylations will be tested by site- directed mutagenesis of the target proteins. (3) To analyze dynamic properties of kinetochores on microtubules in yeast extracts and to determine how kinetochores affect microtubule dynamics. The extract system was successfully adopted for studies of kinetochore association with, and effects on, microtubules. The kinetochore proteins Mtw1 and Spc105 associate with microtubules in the assay. Moreover, they are directionally transported toward microtubule plus ends, and maintain association with disassembling microtubules, all in a cell cycle-dependent manner. This assay will allow mechanisms for kinetochore attachment to MTs, kinetochore activities on MTs, and kinetochore regulation to be revealed.